Enhancement of Immune Response to Vaccine by Interferon Alpha

ABSTRACT

Exogenous cDNA capable of expressing interferon″ activity, exogenous interferon″ protein, inducers of endogenous interferon″ protein activity, inducers of endogenous interferon $ protein activity, inducers of endogenous interferon′ activity, or inducers of other immune-enhancing activity can be combined with a vaccine to enhance an immune response. Specifically disclosed are adjuvant and vaccine combinations where the adjuvant comprises a cDNA capable of expressing interferon″ activity, a complex comprising polyriboinosinic-polyribocytidilic acid, or a complex comprising polyriboinosinic-polyribocytidilic acid, poly-L-lysine, and carboxymethylcellulose and where the vaccine is a live vaccine virus derived from a virus causing porcine reproductive and respiratory syndrome disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser.No.10/416,315, filed Oct. 3, 2003, which is the National Stage ofInternational Application No. PCT/US01/50189, filed Nov. 9, 2001, whichclaims the benefit of U.S. Provisional Application No.60/247,289, filedNov. 9, 2000, all of which are hereby incorporated by reference to theextent not inconsistent with the disclosure herewith.

FIELD OF THE INVENTION

This invention relates to vaccine enhancement particularly for use inpigs and more particularly for porcine reproductive and respiratorysyndrome (PRRS).

BACKGROUND OF THE INVENTION

There have been considerable efforts to identify substances thatpotentiate an immune response to a vaccine. For example, the cytokineinterleukin-12 (IL-12) was reported to be useful as an adjuvant in U.S.Pat. No. 5,976,539, where the IL-12 was provided as a protein ornucleotide coding sequence. Interferon alpha is another cytokinereported to be useful as an oral vaccine adjuvant in, e.g., U.S. Pat.No. 4,820,514. In this patent only the protein form of the cytokine wasused. Furthermore, oral administration of the protein resulted in asystemic effect, and too low or too high a dose of the protein wasundesirable. It would be advantageous to generate a local adjuvanteffect that does not require oral route administration of the proteinand is not so dependant upon dose level.

An important aspect of the development of an adjuvant is the two-foldbreadth potential for its application. Such breadth concerns both theunderlying immune system and the target of the immune system's defense.An adjuvant can be useful in many mammalian species, if the adjuvant hasthe capacity to influence common attributes of the immune systems ofthose species. Further, if the targets of the immune system analogouslyhave common attributes in how they are susceptible to immune responses,then an adjuvant stimulating an immune response should have broadapplicability in defending against a variety of targets.

A need is recognized in the art for enhanced veterinary vaccines. Anarea of significant need for enhanced vaccines is manifested byoutbreaks of porcine reproductive and respiratory syndrome (PRRS). PRRShas a severe impact on the health and reproductive ability of swine. Toaddress this problem, vaccines were developed for the etiological agentof PRRS as reported in U.S. Pat. Nos. 5,989,563 and 6,042,830. In thesepatents, a viral agent capable of causing PRRS disease was isolated andused to develop a modified-live virus (MLV) vaccine. Classically,modified-live virus vaccines are derived from virulent strains that aremodified by techniques such as growth in vitro, with or withoutalteration of conventional in vitro conditions, passage in non-naturalhost cells, or a combination of those techniques. In the U.S. Pat. Nos.5,989,563 and 6,042,830 patents, the reported parental virulent porcineviral isolate is ATCC-VR2332. From this isolate, a modified vaccinevirus was generated by attenuation through multiple passages of growthin vitro in simian cell culture. Thus ATCC-VR2495 was reported to be asuitable MLV for use in vaccine formulations for commercial purposes.

Currently the PRRS vaccines, even those that are of the MLV type, do notconfer adequate protection from disease. In a study designed to test theefficacy of PRRS vaccines, currently available products failed tocompletely protect pigs from clinical disease caused by certain virulentstrains of PRRS virus. Osorio et al., 1998. It was speculated thatdeficiencies in the pig's ability to mount cellular immune responses toPRRS virus may promote the evolution of more virulent strains inaddition to predisposing the animal to virus infection. Alternatively,it was suggested that the PRRS virus itself may be responsible foraltering the cellular immune response. Osorio et al., 1998.

Although the mechanisms that mediate protective immunity against PRRSvirus are unknown, attempts have been made to study the characteristicsof immunity induced by either infection with wild-type PRRS virus orvaccination with a commonly used PRRS modified live virus (MLV) vaccine.An example of PRRS MLV is described in U.S. Pat. Nos. 5,989,563 and6,042,830. Exposure of an animal to either the wild-type virus or theMLV form of the PRRS virus does not stimulate a strong viral purgingimmunity. Virus-specific T cells secreting interferon gamma and virusneutralizing antibodies, both of which have the potential to mediateviral purging, are detected only several weeks after exposure of pigs toPRRS virus. One possible explanation for the failure of PRRS virus tostimulate the development of a strong viral purging immunity is thatPRRS virus, in contrast to other viruses, is a poor stimulator ofinterferon alpha (abbreviated IFNalpha or IFNα) production in swine.Albina et al., 1998. Buddaert et al., 1998. Van Reeth et al., 1999.Consistent with this explanation is a reference indicating that IFNα canaffect the development of anti-viral T cell interferon gamma (IFNgammaor IFNγ responses and peak anti-viral immune defenses. Cousens et al.,1999.

There is considerable evidence in the art, however, that mereaugmentation of IFNα in conjunction with wild-type PRRS virus infectionor PRRS MLV exposure would be expected to have no significant positiveeffect on an immune response. For example, while the importance of IFNαon IFNγ response was shown in the mouse, it is unknown whether a similarresponse would occur in the pig. If the level of IFNα in the pig isindeed unnecessary for the induction of an adequate IFNγ response, thenadditional IFNα present in the pig would have no effect on immuneresponse. Furthermore, while it is believed that PRRS virus can avoidthe effects of the interferon system by blocking the production of IFNor by inhibiting its effects, it is not known which method of avoidance,blocking production or inhibition of effects, is significant or whetherboth methods function in a given organism. Albina, 1998. The lack ofunderstanding about how PRRS directly or indirectly affects the innateIFNα response also makes it unclear that any attempts to further induceendogenous IFNα, to add IFNα (exogenous), or to add IFNα cDNA to expressIFNα would enhance the immune response to PRRS. For example, if PRRSalters an IFNα response by inhibition of IFNα expressed protein, then itwould be expected that no attempt to boost the level of IFNα wouldsucceed in enhancing the immune response to PRRS. In fact, until now ithas appeared unlikely that boosting the level of IFNα in combinationwith PRRS MLV exposure would have any clear effect on enhanced immunity.The present invention makes the unexpected discovery that boosting thelevel of IFNα in an animal can indeed yield enhanced immunity.

The fact that IFNα can enhance immunity coupled with any live virusvaccine is particularly surprising in light of the normal function ofIFNα. Since IFNα is known to have a negative effect on the process ofviral replication, one might reasonably expect that an immune responsefrom exposure to a live vaccine virus would be negatively affected by aboosted level of IFNα.

Even more strikingly, evidence indicates that a heightened level of IFNαcould in fact contribute to a disease state in an infected or exposedanimal. For example, the proinflammatory cytokines IFNα, tumor necrosisfactor alpha, and interleukin-1 have been shown to play key roles inseveral respiratory disease conditions. Van Reeth, 2000. From studiesinvolving various porcine virus infections, it was proposed that therelatively low IFNα response following PRRS infection is related to thelack of acute respiratory disease, severe lung necrosis, andinflammation. Van Reeth, 1999. According to Van Reeth, the absence of aparticular ‘cytokine combination’ such as IFNα, TNFa, and IL-1 duringPRRS infection may in part explain the mild respiratory pathology andthe absence of respiratory disease. As a corollary to this proposal, theaugmentation of IFNα exogenously or endogenously, is predicted tocontribute to a more severe disease state rather than to enhanceimmunity.

Prior research also reveals that at least three other cytokines, IFNγ,IL-12, and IL-18, share the capacity of type one interferons (includingIFNalpha and IFNbeta) to augment immunity by inducing strong T cellproliferation under in vivo conditions. Sprent et al., 2000. Given that“the mechanisms involved here are still unknown,” it is unexpected thata single cytokine such as IFNα serves to enhance an in vivo immuneresponse, if there are multiple independent pathways for achieving asimilar result. Currently, “the biological significance” of at least onetype of immune enhancement, T-cell proliferation, “induced by type 1interferons and other cytokines in vivo is still unclear.” Sprent etal., 2000.

Non-human animal interferons are described in both protein form, U.S.Pat. No. 5,831,023, and in cDNA form (cDNA that expresses IFNα), U.S.Pat. No. 5,827,694. While these references disclose potential use ofIFNα protein, including IFNα expressed from cDNA, in pharmaceuticalcompositions for prophylactic or therapeutic treatment of non-humananimals, the particular use of IFNα in combination with a live virusvaccine is not disclosed and would be disfavored by those skilled in theart for the reasons discussed herein.

Substances capable of inducing endogenous interferon have beenidentified. Levine, 1970. These substances include live viruses witheither DNA or RNA genomes, double stranded RNA, DNA from protozoanparasites, bacterial endotoxin, mannan, mitogens (phytohemagglutinin,streptolysin O, and poke-week mitogen), statolon, helenine, andsynthetic polyribonucleotides.

Other inducers of interferon have been identified. In U.S. Pat. No.5,730,971, the substances flavin adenine dinucleotide, flavin adeninemononucleotide, and riboflavin (vitamin B2) are disclosed as potentialcontributors to the potentiation of an interferon response.

More recently, an adjuvant role for certain short bacterialimmunostimulatory DNA sequences was proposed due to their ability tostimulate T helper-1 responses in animals vaccinated with geneticversions of antigens. Roman M et al., 1997, Nature Med 3:849. These DNAsequences are known to contain CpG (Cytosine-Guanine) motifs that arebelieved to be significant in the immunostimulatory capacity. However,such sequences are suggested for use in vaccine compositions where thevaccine component or subunit thereof is inactivated and not for use withlive organisms such as viruses or modified live viruses.

Inducers of interferon were reported as having potential for resistingviral infection and for treating viral diseases as disclosed in U.S.Pat. Nos. 4,124,702 and 4,389,395. In U.S. Pat. No. 4,124,702, complexesof polymers are reported for induction of interferon production. Thepolymers can be synthetic homopolynucleotides such as polyriboinosinicacid and polyribocytidylic acid mixed in a 1:1 molar ratio (polylC). Forexample, a modified polylC complex was reported to induce seruminterferon in primates in a fashion superior to polylC. Levy, 1975. InU.S. Pat. No. 4,389,395, complexes comprising polylC, poly-L-lysine andcarboxymethylcellulose are reported for use in the induction ofendogenous interferon; these complexes are referred to as polylCLC. BothpolylC and polylCLC were assessed for their ability to induce interferonresponses in pigs. Loewen, 1986; Loewen, 1988. Jordan, 1995. It isbelieved that the polylC and polylCLC provide preferential enhancementof cell-mediated versus humoral immunity. Alternatively, the benefits ofpolylC and polylCLC is believed to be the result of quantitativeimprovement, for example in increases of the numbers of activated cellsor the amount of antibody produced. These and other possiblenon-exclusive mechanisms are ways that interferon inducers can aid inimmunity.

The present invention provides methods and compositions that enhance theefficacy of vaccines, particularly modified live viruses (MLV). Inparticular, the invention enhances the immunity in pigs compared withthe immunity achieved by vaccination with PRRS MLV alone. The ability toenhance immunity is shown by combining a vaccine with any of threeadjuvants: IFNα protein, IFNα cDNA, and inducers of endogenous IFNαproduction.

SUMMARY OF THE INVENTION

The invention comprises compositions and methods for enhancing theimmune response of an animal to a viral vaccine. The inventive methodcomprises administration of a therapeutic composition that enhances thelevel of one or more interferons, particularly interferon alpha, in ananimal that is being treated with the viral vaccine. The level ofinterferon in the treated animal is enhanced, at least locally incertain tissues or cells, over levels of the interferon in the untreatedanimal to enhance the immune response of the animal to a viral vaccine.The therapeutic composition that enhances interferon level preferablycomprises a nucleic acid from which an interferon functional in ananimal can be expressed in cells or tissue of the animal, a materialthat can induce or enhance interferon expression in cells or tissue ofthe animal, or both in a pharmaceutically acceptable carrier. Thistherapeutic composition acts as an adjuvant for enhancing the immuneresponse of the animal to a viral vaccine. The viral vaccine and thetherapeutic composition of this invention are preferablycontemporaneously administered to the animal. The therapeuticcomposition of this invention can also comprise one or more knownadjuvants other than interferon for enhancing immune responses.

In one embodiment, the amount of expressible nucleic acid encoding aninterferon in the therapeutic composition is sufficient to facilitateexpression of encoded interferon in animal cells or tissue andpreferably is present in the composition in an amount sufficient toexpress encoded interferon in animal cells or tissue at a level thatcauses an enhancement of immune response of the animal to a viralvaccine. The amount of a material that induces or enhances interferonexpression present in the composition is sufficient to induce or enhanceexpression of endogenous interferon or to induce or enhance expressionof exogenously administered expressible nucleic acid encoding interferonin cells or tissue of the animal.

The invention provides methods and compositions for enhancing theimmunity of an animal to a virus, particularly a modified live virus byadministering to the animal a vaccine for the virus, and administeringto the animal a therapeutic composition of this invention comprising aninterferon and/or an interferon inducer compound in an amount orcombined amount sufficient to enhance the immunity of the animal to thevirus provided by administration of the vaccine alone. In one aspect ofthis method, an enhanced immune response is assessed by stimulation ofIFNgamma.

The invention also provides methods and compositions for conferringprotective immunity against clinical disease. In a specific embodiment,the methods and compositions of the invention confer partial or completeprotection against PRRS.

In one embodiment, the vaccine and the immunity enhancing interferonand/or interferon inducer compound are administered subcutaneously atthe same time (i.e., within about 1 hour to several hours of eachother.)

The therapeutic compositions and methods of this invention areapplicable for any viral vaccine, for any species of animal, for anyhomologous source of nucleic acid encoding interferon, includinginterferon alpha or beta that is functional in the given species ofanimal, and for any material that induces or enhances expression of aninterferon in the animal. In some cases, heterologous sources of nucleicacid can suffice as a substitute for a homologous source. As anonexclusive heterologous example, a murine source of a nucleic acidencoding IFNα or other interferon can be used in conjunction with anylive virus vaccine, or more specifically a PRRS MLV vaccine, foradministration to a porcine species. As another nonexclusive example, aporcine source of IFNα or nucleic acid encoding another interferoncoding nucleic acid sequence can be used in conjunction with any livevirus vaccine where the vaccine and porcine coding sequence for IFNα orother interferon are administered to a heterologous animal species,e.g., any ungulate.

The therapeutic compositions and methods of this invention areapplicable for arteriviridae and other related viruses. Examples of suchviruses include, but are not limited to, equine arteritis virus, simianhemorrhagic fever virus, and PRRS virus.

Nucleic acids encoding the interferon, e.g., alpha and beta, can benatural or synthetic. The natural or synthetic nucleic acids can betruncations of natural forms or derivatives of natural forms containingbase changes that encode protein variants (or truncations) withconservative amino acid changes compared to a natural form of a givenspecies or type of interferon. Any useful nucleic acid truncations orderivatives encode a protein or polypeptide that substantially retainsthe activity of a given species or type of interferon. Nucleic acidsencoding an interferon are expressible in cells or tissue of an animalto which the expressible nucleic acids are delivered. Expressiblenucleic acid sequences are under the regulatory control of regulatoryelements including a promoter, polyadenylation signal sequence andoptionally other related sequences that control expression or expressionlevels of the nucleic acid sequence in a cell or tissue of a givenanimal. The promoter employed may be constitutive or may be directselective expression in a selected tissue or in a selected environment.An expressible nucleic acid, as used herein, comprises any nucleic acidsequences necessary for expression of the interferon coding sequenceoperably linked to that coding sequence for expression in a cell ortissue of an animal. The expressible nucleic acid encoding an interferoncan be provided in an expression vector, such as a plasmid, whichoptionally contains other expressible coding sequences, such asselectable or detectible markers.

Regulatory sequences in the expressible nucleic acid can be heterologousor homologous to the nucleic acid encoding interferon, e.g., theexpressible nucleic acid can contain a polyadenylation sequence of thenatural interferon coding sequence or a polyadenylation sequenceobtained from another source, i.e., from another gene. The regulatorysequences may be obtained from nucleic acid of the same species as theinterferon coding sequences or from nucleic acid of a different species,e.g., a polyadenylation sequence from a bovine gene can be employed withan interferon coding sequence of a porcine animal.

In particular, the invention comprises compositions and methods forenhancing the immune response of porcine animals to a PRRS vaccine. Thecompositions and methods utilize the ability of exogenous interferoncoding sequences, as administered in expressible nucleic acid, inducersof endogenous interferon activity, e.g., inducers of endogenousinterferon alpha protein activity, inducers of endogenous interferonbeta protein activity, and inducers of endogenous interferon gammaactivity, or mixtures thereof, in combination with a vaccine compositionto enhance an immune response.

In one embodiment, a therapeutic composition comprises an expressiblenucleic acid sequence encoding an interferon alpha and apharmaceutically acceptable carrier. The nucleic acid sequence ispresent in the composition in an amount sufficient to express an amountof interferon alpha sufficient to exhibit enhancement of immune reponseon delivery to an animal, particularly a pig. The composition can beprovided with appropriate components to facilitate delivery andexpression of the nucleic acid in a desired selected organism, such as apig. The composition optionally combines expressible interferon-″nucleic acid sequence with a live virus vaccine, or more specifically aPRRS MLV vaccine. An exemplary interferon-″ nucleic acid sequence isthat of FIG. 1.

In another embodiment, a method is provided for enhancing immunity byadministering a vaccine composition to an animal and administering anadjuvant composition comprising an expressible nucleic acid forinterferon-α to an animal. The order of administration of the vaccinecomposition and the adjuvant composition is preferably contemporaneous,i.e., sufficiently close in time so that the adjuvant results in anenhancement of immune response in the animal. Administration of the twocompositions can be simultaneous or sequential; if administration issequential, either the adjuvant can be administered to the animal firstand the vaccine second, or vice versa. Contemporaneous administrationencompassed administration of the adjuvant up to about one week beforeor one week after administration of the vaccine. Preferably the vaccineand adjuvant are administered to the animal on the same day and morepreferably within several hours of each other.

In another embodiment of the invention, the immunity enhancinginterferon or interferon enhancer molecule is administered to the samelocalized site as the vaccine.

In a specific embodiment, a method is provided for enhancing immunity byfirst administering a vaccine composition to an animal and thereafteradministering the adjuvant composition comprising an expressible nucleicacid encoding interferon alpha. The adjuvant composition is preferablyadministered up to one week after the vaccine is administered.

In another embodiment, a method for enhancing immunity by administeringto an animal a vaccine composition and administering to an animal anadjuvant composition comprising a material capable of inducing an IFNαresponse, IFNβ response, IFNγ response, or other response resulting inenhanced antiviral immunity is provided. The interferon γ response canbe induced by the activity of interferon α, interferon β, or othermediator. In a particular embodiment, the material that induces orenhances interferon activity is a pharmaceutically acceptablecomposition comprising polyriboinosinic-polyribocytidylic acid,poly-L-lysine and carboxymethylcellulose optionally in combination witha pharmaceutically acceptable carrier. In another particular embodiment,said material is a pharmaceutically acceptable composition comprisingpolyriboinosinic acid-polyribocytidylic acid complex optionally incombination with a pharmaceutically acceptable carrier. Alternatively,the material can be any of the following which induce an interferonresponse, particularly an interferon α response in a given animal: liveRNA viruses, Inactivated RNA viruses, Live DNA viruses, Inactivated DNAviruses, DNA from DNA viruses, DNA from bacteria, DNA from bacterialplasmids, DNA from synthetic plasmids, DNA from organisms in the familyRickettsiae, DNA from protozoan parasites, Bacterial endotoxin,Double-stranded RNA, Double stranded synthetic polyribonucleotides;Phytohemagglutinin; Mannan; Streptolysin O, Poke-weed mitogen, Statolon,Helenine, Flavin adenine mononucleotide, Flavin adenine dinucleotide,and Riboflavin.

In yet another embodiment, the invention relates to an adjuvantcomposition comprising an expressible interferon α having the sequenceof SEQ ID NO:3 present in the composition at a level such that the levelof interferon α expressed in an animal to which the composition isadministered is sufficient to enhance immune response to a vaccineadministered to the animal. In a particular aspect of this embodimentthe animal is a pig.

In yet another embodiment, the invention provides a method for enhancingan immune response of an animal to a vaccine comprising the steps ofadministering a vaccine antigen composition to an animal in an amounteffective to stimulate an immune response and administering to theanimal an adjuvant composition comprising nucleic acid having capabilityof expressing interferon activity in an amount effective to potentiatethe immune response to the vaccine antigen composition. In one aspect ofthis method the interferon is a Type 1 interferon, interferon alpha orinterferon beta. In another aspect of the method the interferon isporcine interferon alpha. In another aspect of the method the animalthat is vaccinated is a pig. In yet another aspect of the method thevaccine antigen composition comprises a live vaccine virus. In someaspects of this method the live vaccine virus is derived from an agentcapable of causing clinical signs of porcine reproductive andrespiratory syndrome disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Nucleotide (SEQ ID NO:3) and predicted Amino Acid sequence (SEQID NO:4) of amplified porcine interferon-α cDNA. The nucleotides andamino acids that are divergent from those of sequence in GenBank:X57191are in bold and underlined.

FIG. 2: Schematic representation of plasmid pINA3. The relativepositions and direction of transcription of the cytomegalovirus promoter(CMV), porcine interferon alpha cDNA, bovine growth hormonepolyadenylation signal sequence (BGH polyA), SV40 origin, bacterialneomycin resistance gene, SV40 polyadenylation signal sequence (SV40polyA) and bacterial ampicillin resistance gene are indicated.

FIG. 3: Adjuvant effect of IFNalpha cDNA on IFN-gamma response to PRRSMLV in 8 week old pigs; see Example 2. Adjuvant effect ofinterferon-alpha on the virus-specific interferon-gamma response ofswine to a PRRS modified live virus (MLV) vaccine. Groups of pigs (5 pergroup) were immunized with a commercial PRRS MLV vaccine either alone orwith the simultaneous intramuscular injection of 200 ug of the plasmidpINA3 that contains the cDNA for porcine IFN-alpha. At the indicatedtimes after immunization peripheral blood mononuclear cells wereisolated from those animals and the number of PRRS virus-specificinterferon-gamma secreting cells determined by ELISPOT.

FIG. 4: Adjuvant effect of Poly lCLC on IFN-gamma response of swine toPRRS virus in 6 week old pigs; see Example 4. Kinetics and intensity ofthe PRRSV-specific CMI response following immunization with MLV+polylCLC as adjuvant. Pigs (n=6) were given a secondary immunization at week9. CMI is expressed as group mean+/−SEM. *Denotes that the value issignificantly different from vaccine only group (*p<0.05; **p<0.07).

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein:

-   complementary DNA: cDNA-   Interferon α: IFNalpha or IFNα-   Interferon β: IFNbeta or IFNβ-   Interferon γ: IFNgamma or IFNγ-   modified live virus: MLV-   polyriboinosinic acid and polyribocytidylic acid complex: polylC-   polyinosinic:polycytidylic acid, poly-L-lysine and    carboxymethylcellulose complex: poly lCLC-   porcine reproductive and respiratory syndrome: PRRS

As used herein, the phrase “enhancing an immune response” refers to thestrengthening of an existing immune response to a pathogen and/or theinitiation of an immune response to a pathogen. Those of ordinary skillin the art recognize that there are a variety method for assessing suchenhancements, for example, humoral antibody measurements, cytokinemeasurements, assessing animal health or well-ness, clinical protectionfrom disease, and measuring cell mediated changes in immunity.

As used herein, the term “interferon” generally refers to any of amember of the family of molecules, sometimes referred to as cytokines,that have interferon activity such as modulation of immune response. Theterm “interferon” includes polypeptides and fragments thereof which haveinterferon activity such as chimeric or mutant forms of interferon inwhich sequence modifications are introduced, for example, to enhanceselected characteristics of the molecule, without substantiallyaffecting the nature of the biological activity (see U.S. Pat. Nos.5,582,824, 5,593,667, and 5,594,107 all of which are incorporated byreference to the extent not inconsistent herewith.)

As used herein, the terms “porcine reproductive and respiratorysyndrome” or “PRRS” refers to the causative agent of a disease sometimesreferred to as “mystery swine disease”, “swine infertility andrespiratory syndrome”, and “blue ear disease.” The terms “porcinereproductive and respiratory syndrome” or “PRRS” are intended to includeantigenic, genetic and pathogenic variations among PRRS virus isolates(Wensvoort et al., 1992, J. Vet. Diagn. Invest., 4:134-138; Mardassi etal., 1994, J. Gen. Virol., 75:681-685.)

As used herein a “vaccine composition” or “vaccine antigen composition”is any composition containing a molecule capable of stimulating animmune system response. This invention is particularly related tovaccines that stimulate an immune system response to a virus, and morespecifically to a virus that is associated with a disease state of ananimal. Vaccines for PRRS are of particular interest. The compositionsand methods of this invention particularly relate to vaccinecompositions that comprise modified live virus (MLV) and in this regardare particularly directed to vaccines that comprise an MLV of PRRS.Vaccine compositions may contain other ingredients as known in the artto facilitate or benefit functionality. Vaccine dosage levels for agiven application can be determined by well-known methods.

As used herein an “adjuvant” is a molecule capable of enhancing animmune system response to a vaccine. In this invention an adjuvantcomposition can comprise a nucleic acid capable of expressing aninterferon, particularly an interferon α. Interferon generated byexpression from the exogenously administered nucleic acid sequencefunctions, alone or in combination with interferon generated byexpression from endogenous nucleic acid sequences native to an animal,to enhance immune response to a vaccine that is administered to theanimal. Interferon can directly or indirectly facilitate immuneenhancement; for example, the interferon expressed from exogenouslyadministered nucleic acid can induce or activate one or moreintermediate species which in turn facilitates immune enhancement. Inthis invention an adjuvant composition can alternatively comprise amaterial that induces or enhances the activity of interferon,particularly interferon α. This material can function to induce orenhance the activity of interferon generated from exogenouslyadministered expressible nucleic acid or that generated from endogenousnucleic acids native to the animal. The material can function directlyto induce or enhance interferon activity or indirectly by induction orenhancement of the activity or expression of an intermediate species.The material may function to induce or enhance expression levels of aninterferon or may otherwise enhance or activate interferon forenhancement of immune response. The material is present in the adjuvantcomposition at a level sufficient to enhance an immune response to avaccine administered to an animal. Enhancement of immune response by anadjuvant of this invention is measured as any statistically significantincrease in immune response compared to control response in the absenceof the adjuvant as evaluated by any method accepted in the art. Adjuvantcompositions may contain other ingredients as known in the art tofacilitate delivery of an expressible nucleic acid to a cell or tissuefor expression or facilitate delivery of the interferon inducer orenhancer to an appropriate cell or tissue. Dosage levels of adjuvant canbe determined by well-known methods.

An adjuvant composition of this invention can comprise both a nucleicacid capable of expressing an interferon as well as a material that caninduce or enhance activity of interferon. In this case the combinedamounts of nucleic acid and the interferon inducer or enhancer aresufficient to result in a measurable enhancement of immune response to agiven vaccine.

Preferred adjuvant compositions of this invention are those thatcomprise an expressible nucleic acid encoding an interferon α, amaterial which induces or enhances the activity of interferon α or both.Preferred materials which induce or enhance activity of interferon αinclude poly lC and poly lCLC.

Subunit Polypeptide Vaccines

The present invention also provides for vaccines comprising one or morePRRS virus or arterivirus polypeptides. A nucleic acid encoding such apolypeptide or polypeptides is constructed in a vector suitable for aprokaryotic or eukaryotic host and capable of expressing one or morePRRS virus polypeptides. Examples of such subunit/polypeptide PRRSvaccines are given in U.S. Pat. No. 6,251,397 to Paul et al. which ishereby incorporated by reference to the extent not inconsistentherewith. There are numerous Escherichia coli expression vectors knownto one of ordinary skill in the art useful for the expression of theantigen. Other microbial hosts suitable for use include bacilli, such asBacillus subtilus, and other enterobacteriaceae, such as Salmonella,Serratia, and various Pseudomonas species. In these prokaryotic hostsone can also make expression vectors, which will typically containexpression control sequences compatible with the host cell (e.g., anorigin of replication). In addition, any number of a variety ofwell-known promoters will be present, such as the lactose promotersystem, a tryptophan (Trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters willtypically control expression, optionally with an operator sequence, andhave ribosome binding site sequences for example, for initiating andcompleting transcription and translation. If necessary, an aminoterminal methionine can be provided by insertion of a Met codon 5′ andin-frame with the coding sequence. Also, the carboxy-terminal extensionof the sequence can be removed using standard oligonucleotidemutagenesis procedures.

Alternative vectors for the expression of PRRS virus polypeptides ineukaryotic hosts, e.g., mammalian or porcine cells, similar to thosedeveloped for the expression of human gamma-interferon, tissueplasminogen activator, clotting Factor VIII, hepatitis B virus surfaceantigen, protease Nexinl, and eosinophil major basic protein, can beemployed. Further, the vector can include cytomegalovirus promotersequences and a polyadenylation signal available for expression ofinserted DNAs in eukaryotic cells.

Vaccine Composition and Carriers

Compositions can be administered alone or in combinations, e.g., as acomplex with cationic liposomes, encapsulated in anionic liposomes,enclosed in chochleates, or they can be encapsulated in microcapsules.Compositions can include various amounts of the selected composition incombination with a pharmaceutically acceptable carrier and, in addition,if desired, may include other medicinal agents, pharmaceutical agents,carriers, adjuvants, diluents, etc.

Any vaccine composition of this invention can comprise apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier in the vaccine of the instant invention can comprise saline orother suitable carriers (Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRCPress, Inc., Boca Raton, Fla., 1987). An additional adjuvant can also bea part of the carrier of the vaccine, in which case it can be selectedby standard criteria based on the antigen used, the mode ofadministration and the subject (Arnon, R. (Ed.), 1987). The DNA vaccinescan be incorporated in liposomes or chocleates to enhance in vivotransfection. Genetic adjuvants, such as immunostimulatory sequences(ISS) and cytokine-encoding nucleic acids, can also be employed. SeeHorner A A et al., 1998, Immunostimulatory DNA Is a Potent MucosalAdjuvant, Cellular Immunology 190:77-82, and Roman M et al., supra.

Additional Adjuvants

The compositions and methods of the invention can be administered withan additional adjuvant. In general, one or more than one additionaladjuvant can be used to enhance the activity of the invention as long asa composition enhances the immune response to a vaccine or at least doesnot substantially inhibit the immune response. Examples of suchadjuvants are found in “Vaccine design: the subunit and adjuvantapproach” eds. Michael F. Powell and Mark J. Newman PharmaceuticalBiotechnology v. 6, Plenum Press 1995, New York, see e.g., chapter 7 “Acompendium of Vaccine Adjuvants and Excipients” by Frederick R. Vogeland Micheal F. Powell and chapter 29, “cytokine-containing liposomes asadjuvants for subunit vaccines” by Lachman et al., which is herebyincorporated by reference. Examples of additional adjuvants that can beused in the invention include, but are not limited to, cytokines e.g.,IL-2, IL-12, and other cytokines, cytokine-containing liposomes, alum(aluminum hydroxide), aluminum phosphate, and calcium phosphate.

Routes and Methods of Administration

Commercially available PRRS MLV currently indicates that administrationof the vaccine should be performed parenterally by intramuscularinjection using a conventional needle and syringe. However, embodimentsof the present invention are not necessarily restricted to such route ormethod.

Vaccine compositions, including interferon-alpha and other materialslike those that are inducers of interferon can be administered to asubject by any of many standard means for administering the particularcomposition. For example, compositions can be administered orally,sublingually, intraocularly, intranasally, intravenously, byintramuscular injection, intradermally, by intraperitoneal injection,topically, transdermally, and the like. Parental administration, ifused, is generally characterized by injection. Injectables can beprepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions.

Other methods for delivery can include formulation with cationic lipidsand liposomes; this can be applicable to either the DNA form or proteinform of a cytokine adjuvant or to a chemical such as one capable ofimmune stimulation, for example by induction of an endogenous cytokine.See Pachuk C J et al., 2000, Curr Opin Mol Ther Apr 2(2):188-98; VanSlooten M L et al. 2001, Biochim Biophys Acta 1530:134-45; Van Slooten ML et al., 2000, Pharm Res 17:42-48; Lachman L B et al., 1996, EurCytokine Netw 7:693-8. Further methods for delivery can includeelectroporation, cationic microparticles, ultrasonic distribution, andbiolistic particle delivery techniques.

An embodiment of the invention comprises a mammalian expression vectorcontaining porcine IFN-α cDNA. A particular embodiment comprises cDNAencoding porcine IFN-α was prepared by RT-PCR using RNA isolated frompig lymphocytes previously infected with pseudorabies virus (tostimulate IFN-α production). Primers were designed based on thenucleotide sequence of porcine IFN-α cDNA (Lefevre and La Bonnardiere1986). Products of the anticipated size (590 bp) were cloned into thepCR®2.1 plasmid (Invitrogen Corp., Rockville, Md.), and an insert havingthe predicted restriction enzyme sites was sequenced. A comparison ofthe amplified sequence to the one previously reported revealed threenucleotide differences within the coding sequence. These differencesresulted in two amino acid changes. The IFN-α cDNA was excised from therecombinant pCR®2.1 plasmid and placed under the transcriptionalregulation of the cytomegalovirus promoter in pcDNA3 (Invitrogen Corp)to generate pINA3. To verify that an active cytokine was encoded by theamplified cDNA, Chinese hamster ovary (CHO) cells were transfected withpINA3 and single cell clones resistant to genticin were prepared.Supernatants from the clones were tested for the ability to inhibit thereplication of an interferon-inducer negative strain of vesicularstomatitis virus in Madin Derby bovine kidney (MDBK) cells. Clonesproducing from 0 to greater than 200,000 units (1 unit inhibits 50% ofVSV replication) of IFN-α were detected.

Another embodiment comprises a preparation of a chemical compound,polylCLC. In another particular embodiment, the following chemicals areobtained from Sigma-Aldrich (St. Louis, Mo): Poly-L-Lysine (Cat. No.P0879), poly lC (Cat. No. P0913), and carboxymethylcellulose, lowviscosity (Cat. No. C5678). Poly lC (500 ml; 4.0 mg/ml); poly-L-lysine(250 ml; 6.0 mg/ml); and 2% carboxymethylcellulose (250 ml) wereprepared in pyrogen-free 0.85% NaCl. Poly lCLC (stabilizedpolynucleotide) was prepared following the method of Levy, Baer et al.(1975) with minor modifications. Poly l:C was re-annealed by heating at71° C. for 1 hour and cooled slowly. Annealed poly l:C was then mixedwith equal volumes of 6.0 mg/ml poly-L-lysine in normal saline and 2%carboxymethylcellulose . The final concentration of poly l:C was 1mg/ml. This preparation was stored at 4° C. until needed.

THE EXAMPLES Example 1 Challenge Study of PRRS MLV Vaccine ComprisingIFN″ cDNA Adjuvant

Enhanced immunity is achieved by supplementing a vaccine with a cDNAmolecule capable of expressing interferon α. In an embodiment of theinvention, Ingelvac ® PRRS MLV is used with cDNA encoding porcine IFNαand capable of expressing IFNα. The Ingelvac PRRS MLV correspondsapproximately to ATCC-VR2495, supra. The reproductive efficacy of PRRSMLV with porcine IFNα cDNA is measured relative to that of PRRS MLValone via a clinical study. The key parameter of efficacy measured inthe study is the number of piglets surviving through 28 dayspost-farrowing which were born to vaccinated female pigs, gilts, or sowsas compared to the results for unvaccinated animals following PRRS viruschallenge of both vaccinated and unvaccinated animals at about 90 daysof gestation.

In a preferred embodiment of the invention, the cDNA encoding porcineIFNα is prepared as follows:

A cDNA copy of porcine interferon-α mRNA was prepared by the use of areverse transcription-polymerase chain reaction (RT-PCR) in thefollowing manner. Forward (INF-AF; SEQ ID NO:1; TCTGCAAGGTTCCCAATG) andreverse (INF-AR; SEQ ID NO:2; GTCTGTCACTCCTTCTTCCTG) primers weredesigned based on the sequence of the porcine interferon-α genepresented in GenBank accession number X57191. The INF-AF primer isidentical to the initiation codon of the porcine interferon-α transcriptand the fifteen, upstream nucleotides whereas the complement of thetranslation termination signal and flanking sequences resides withinprimer INF-AR. The reverse primer was used to direct synthesis of cDNAby Superscript II reverse transcriptase (Life Technologies, GrandIsland, N.Y.) from total RNA, isolated from pig leukocytes previouslyinfected in vitro with pseudorabies virus (to stimulate production ofinterferon-α). The resulting products were amplified using the HighFidelity PCR System (Boehringer-Mannheim, Indianapolis, Ind.) and bothINF-AF and INF-AR primers. An amplicon of the predicted size (590 bp)was purified by agarose gel electrophoresis and subsequently cloned intothe TA vector, PCRII (Invitrogen, Carlsbad, Calif.), to create pSIN3.

Initial indication that pSIN3 contained the porcine interferon-α cDNAwas based on the demonstrated presence of predicted restrictionendonuclease sites within the insert. A comparison of the nucleotidesequence of the cloned amplicon (FIG. 1) to that described in theGenBank submission showed 99% identity with only three mismatches. Thesewere a G to C, A to G, and G to T change at positions 327, 341, and 437,respectively. The nucleotides and amino acids that are divergent fromthose of sequence in GenBank: X57191 are in bold.

These different nucleotides resulted in no change in primary structure:a non-conservative replacement of tyrosine by cysteine, and anon-conservative replacement of arginine by leucine, respectively (FIG.1). Whether these alterations are due to amplification errors,polymorphisms in the interferon-α gene, or to amplification of atranscript arising from a non-allelic interferon-α gene (there are atleast eleven) is unclear.

To determine whether the amplified porcine interferon-α cDNA encoded anauthentic cytokine, the intact cDNA was excised from pSIN3 by digestionof the plasmid with Hind III and Xho I and inserted into the respectivesites of the eucaryotic expression vector pcDNA3 (Invitrogen) to createpINA3 (FIG. 2). In FIG. 2, the relative positions and direction oftranscription of the cytomegalovirus promoter (CMV) porcine interferonalpha cDNA, bovine growth hormone polyadenylation signal sequence (BGHpolyA), SV40 origin of replication, bacterial neomycin resistance gene,SV40 polyadenylation signal sequence (SV40 polyA) and bacterialampicillin resistance gene are indicated.

In this plasmid, expression of the inserted porcine interferon-α cDNA isregulated by the cytomegalovirus enhancer-promoter and the resultingtranscripts are polyadenylated due to a downstream bovine growth hormonepolyadenylation signal sequence. Chinese hamster ovary (CHO) cells werethen transfected with pINA3 and 48 hr later the overlaying medium wasassayed for the presence of porcine interferon-α in the following ways.First, the ability to inhibit vesicular stomatitis virus (VSV)replication in Madin-Darby bovine kidney (MDBK) cell monolayers wasexamined. Second, the ability to stimulate porcine leukocytes to produceinterferon-γ was tested. Since positive results were obtained only whenusing the supernatants from pINA3-transfected cells, production of abiologically active cytokine was evident.

The PRRS vaccine comprises any effectively attenuated form of PRRS. Aparticular example is a serial or lot of Ingelvac PRRS® MLV manufacturedaccording to the commercial outline of production and used per labeldirections. The experimental material is a similar PRRS vaccine to whicha quantity of nucleic acid vector containing expressible cDNA forporcine IFNα is added, in a range of 0.1 to 1000 μg per kg of bodyweight. Alternatively, the experimental material is the PRRS vaccine andthe quantity of IFNα nucleic acid which are not mixed together butdelivered to the test subject separately.

In a preferred embodiment, the test subjects are a commercial crossbreed of female porcine animals of an age used in breeding operations.The gilts or sows are about at least 6 months of age and ofapproximately uniform weight for age. The animals are confirmed ashaving naive serological status with respect to PRRS as measured byIDEXX PRRS ELISA (Westbrook, Me.), a commercially available test forporcine antibody specific to PRRS antigens. The gilts or sows arevaccinated with a composition of the invention from about 4 weeks priorto 1 week prior to breeding, with or without previous vaccination, andthe animals are not exposed to either the vaccine composition or theinterferon alpha adjuvant component during gestation.

In another embodiment, the test subjects are pigs about at least 3 weeksof age at the time of vaccination and may or may not receive a secondvaccination prior to breeding.

In yet another embodiment, the females are confirmed as pregnant byultrasound and are vaccinated with a composition of the invention atfrom about 30 days to about 45 days of gestation at the time ofvaccination.

In a particular embodiment, the clinical study includes four test groupsof 8 female breeding animals per group. Group 1 receives PRRS MLV on Day0, equivalent to about 30 days of gestation. Group 2 receives PRRS MLVsupplemented with cDNA for porcine IFNα. The inoculation of animals withchallenge virus occurs on about Day 60, the equivalent of about 87 to 92days of gestation. Group 3 animals are not vaccinated but arechallenged. Alternatively, Group 3 animals are treated with the invertedcoding sequence of IFNalpha as a control and then challenged. Group 4animals are not vaccinated and not challenged. The farrowing performanceis evaluated by observing the health status of the piglets born.

The challenge material is a virulent strain of PRRS virus, strain17198-6. The challenge material is diluted in Modified Eagles Mediumsupplemented with 4% fetal calf serum such that the concentration isabout log10 3.5+/−0.5 per ml. The challenge dose is administered bygiving a total of 2 ml intranasally, one ml per nostril. The intranasaladministration is performed using 2 ml of challenge material and asyringe with 16 gauge catheter tubing. The challenge strain can bederived from any virulent PRRS virus such as a field isolate. Anotherexample of another suitable challenge strain is ATCC VR-2385(Thanawongnuwech, R. et al. 1997 Vet. Immunol. Immunopathol.59:323-335.)

Following challenge, multiple analyses are performed includingevaluation of serological data, viremia data, body temperature data,clinical observations, and farrowing results. The transplacentaltransmission of PRRS virus after administration of the virus isevaluated. The farrowing performance of the sows is the main criterionused to determine efficacy of the test articles. Specifically, thenumber of mummies versus stillborn fetuses (autolyzed in utero deathversus fresh-dead) are compared, and parameters including the healthstatus and performance of the pigs born alive through 28 days postfarrowing are monitored.

The results between and among groups are compared to determine thedegree of immune enhancement achieved by immunization with PRRS MLValone versus immunization using PRRS MLV and cDNA for porcine IFNα.

In another embodiment, one or more than one vaccination (administration)of PRRS MLV is given in conjunction with one or more than oneadministration of cDNA for porcine IFNα where the vaccination andadministration can occur at the same or different times. Theadministrations of vaccine and adjuvant, if separate, are preferablycontemporaneous, i.e., administered within a time span that issufficiently short that the immune enhancement can occur, preferablywithin about one week of vaccination. Substantially simultaneousadministration (separate administrations of vaccine and adjuvant) withrapid sequential administration or co-administration of the vaccine andadjuvant can be employed. Alternatively, separate administration ofvaccine and adjuvant can occur at intervals up to about one week.

Example 2 Adjuvant Effect of IFNalpha cDNA on IFNgamma Response to PRRSMLV

Eight-week-old Yorkshire×Landrace cross-bred pigs were obtained from anunvaccinated, specific-pathogen-free (SPF) facility at the University ofIllinois Veterinary Research Farm. The pigs were randomly segregatedinto 3 groups. One group (n=5) of pigs was immunized intramuscularly(I.M.) into the adductor muscle (inner thigh) with 2.0 ml of PRRS MLVvaccine (Ingelvac PRRS MLV; Nobl). A second group of pigs (n=5) wasvaccinated with the same MLV vaccine immediately followed by anintramuscular injection at an adjacent site of 200 μg of a plasmidcontaining the cDNAs for the porcine IFN-α. A third group (n=2 pigs)remained as unvaccinated negative controls that were maintained in aseparate room. Peripheral blood mononuclear cells were isolated weeklyand used to monitor the virus-specific immune response to the PRRS virusvaccine. The number of PRRS virus-specific IFN-gamma-secreting cells wasmeasured using an ELISPOT assay (as described in Zuckermann, F. A.,Husmann, R. J., Schwartz, R., Brandt, J., Mateu de Antonio, E. AndMartin, S. 1998. Interleukin-12 enhances the virus-specific interferongamma response of pigs to an inactivated pseudorabies virus vaccine.Vet. Immunol. Immunopath. 63:57-67).

The accompanying figure (FIG. 3) shows the enhancing effect of IFN-alphaon the virus-specific IFN-g response to vaccination against PRRSV.Groups of pigs (n=5) were immunized with a commercial PRRSV modifiedlive virus vaccine given either alone (open squares) or in combinationwith the simultaneous intramuscular injection 200 μg of a plasmidcontaining the cDNA for porcine IFN-alpha (open triangles). A controlgroup remained as unvaccinated negative controls (asterisk). At theindicated times peripheral blood mononuclear cells were isolated fromthese animals and tested in an IFN-gamma ELISPOT assay as described(Zuckermann et al., 1998). The IFN-gamma response is expressed as thegroup mean frequency of virus-specific IFN-g-secreting cells/10⁶PBMC±standard error of the mean.

Example 3 Performance Study of PolylCLC Adjuvant

Enhanced immunity was achieved by supplementing a vaccine with aninducer that induces or enhances endogenous IFNα activity, endogenousIFNβ activity, endogenous IFNγ activity, or other activity that resultsin enhanced immunity. In a preferred form of the invention, the induceris a composition of polylCLC. In another preferred form of theinvention, Ingelvac® PRRS MLV is used with the inducer. Efficacy of theadjuvant is measured as the intensity of the humoral and cellular immuneresponse to the supplemented vaccine, compared to unsupplementedcontrols, as well as the number of piglets surviving through 28 dayspostfarrowing born to vaccinated sows as compared to sows receiving thevaccine without the polylCLC composition or unvaccinated sows.

In a preferred embodiment, the inducer is prepared as follows: theadjuvant composition comprises Polyinosinic:polycytidylic acid complexedwith poly-L-lysine and carboxymethylcellulose (poly lCLC) at a selecteddosage.

The following chemicals are all obtained from Sigma-Aldrich (St. Louis,Mo.). Poly-L-lysine (mol wt, 1,000-4,000 Daltons).Polyinosinic:polycytidylic (poly lC) acid (average mol. Wt.200,000-500,000). Carboxymethylcellulose (low viscosity).

The poly lCLC compound is prepared following the method described byLevy et al. (1975). To prepare poly lCLC the following solutions areprepared in pyrogen-free 0.85% NaCl: 500 ml of poly lC (4 mg/ml); 250 mlof poly-L-lysine (6 mg/ml); and 250 ml of 2% carboxymethylcellulose.Briefly, poly lC is re-annealed by heating at 71° C. for 1 h and allowedto cool slowly is then added to a mixture of equal volumes of thepoly-L-lysine and carboxymethylcellulose solutions to give a finalconcentration of poly lC of 2 mg/ml. The complex is stored at 4° C.

The proposed dose of a poly lC for pigs is about 20 micrograms of thepoly lC/kg of body weight. Thus, for example a pig with a body weight of300 lb would receive 1.5 cc of the poly lCLC solution. This dose isbased on best guess estimate based on literature using this compound inpigs and monkeys.

Smaller molecular weight forms of poly lC can be made or produced uponrequest to a vendor or can be synthesized following literature methods.

As in Example 1, the PRRS vaccine comprises any effectively attenuatedform of PRRS. A particular example is a serial or lot of Ingelvac PRRS®MLV manufactured according to the commercial outline of production andused per label directions. The experimental material is a similar PRRSvaccine to which a quantity of polylCLC in a pharmacuetically acceptablecarrier can be added. The quantity of polylCLC can be in a range of 1 to200 micrograms per kg of body weight. Alternatively, the experimentalmaterial is the PRRS vaccine and the quantity of polylCLC which are notmixed together but delivered to the test subject separately.

The test subjects are a commercial cross breed of female porcineanimals, about 6 to 8 weeks of age and of approximately uniform weightfor age. The animals are confirmed as having naive serological statuswith respect to PRRS as measured by IDEXX PRRS ELISA (Westbrook, Me.), acommercially available test for porcine antibody specific to PRRSantigens.

The clinical study includes three test groups of 10 to 30 animals pergroup. At 6 weeks of age, Group 1 gilts receive PRRS MLV at 6 weeks ofage, and Group 2 gilts receive PRRS MLV and polylCLC . The respectivetreatments (PRRS MLV or PRRS MLV and polylCLC) are repeated 8 weekslater at 14 weeks of age. Group 3 gilts are untreated controls. Allanimals are introduced into the production herd at 26 weeks of age.Farrowing performance is evaluated by observing the health status of thepiglets born.

To evaluate the effectiveness of the vaccine supplemented with polylCLC,the development of humoral and cellular immunity of all pigs ismonitored by sampling peripheral blood at 5, 8, 13, 16, and 18 weeks ofage. The humoral immune response is measured by the IDEXX ELISA test andwith a virus-neutralization test. The cellular immunity is measured withthe IFNg ELISPOT assay developed by Zuckermann. To monitor the effect oftreatment with PRRS MLV and polylCLC, the treated animals are monitoredfor performance including litter size and viability of the piglets. Suchmonitoring occurs for at least one pregnancy. Specifically, the numberof mummies, stillborn fetuses (autolyzed in utero death versusfresh-dead) and the health status and performance of the pigs born alivethrough 28 days postfarrowing is monitored. Gilts and sows are evaluatedfor behavior, respiration, and cough.

At farrowing, general observations include the number of live bornpiglets (weak or healthy), and the stillborn and mummified piglets areregistered. Mummies are measured for crown-rump length at farrowing anda determination is made for time of death during fetal development.Stillborn piglets are classified as autolyed in utero or fresh-dead.Live-born piglets are examined daily from birth through 28 days of agefor general clinical health status. Deaths of live-born piglets arerecorded. Dead piglets are weighed after farrowing and body fluidsamples (abdominal or thoracic cavity or lung lavage) are collected fordetection of PRRS virus. Virus isolation assays are performed on allpresuckle sera obtained from live-born piglets to detect PRRS viremia.At birth and on day 28 of life, all live piglets are weighed todetermine weight gain within the first four weeks of age. Livebornpiglets that die during the study are weighed as soon as possible afterdeath. Clinical observations include behavior, respiration, and coughper raw observations or a scoring system.

Following data collection, analyses are performed including evaluationof serological data, viremia data, body temperature data, clinicalobservations, performance data including weight gain, survival data, andfarrowing results.

The results between and among groups are compared to determine thedegree of immune enhancement achieved by immunization with PRRS MLValone versus immunization using PRRS MLV and polylCLC.

In another preferred form of the invention, one or more than onevaccination of PRRS MLV is given in conjunction with one or more thanone administration of polylCLC, where the vaccination and administrationcan occur at the same or different times. Preferably the intervalsbetween administration of separate vaccine and adjuvant or multipleadministrations of vaccine and adjuvant are less than about one week.

Example 4 Adjuvant Effect of Poly lCLC on IFNgamma Response of Swine toPRRS Virus

As an alternative to a challenge study, the enhancement effect ofvaccine compositions is evaluated by measuring increased levels ofIFNgamma post-vaccination. Twelve 6-week-old Yorkshire×Landracecross-bred pigs were obtained and randomly assigned to 2 groups (n=6).The first group was immunized intramuscularly with 2.0 ml of PRRS MLVvaccine (Ingelvac PRRS MLV; Nobl). The other group was immunized withPRRS MLV vaccine co-administered with 0.25 mg/kg of poly lCLC. This doseof poly lCLC has been shown to induce maximum IFN-α titers in the seraof 10-week-old pigs (see Lowen K. G. and Derbyshire, J. B. 1988.Interferon induction in piglets with polyinosinic:polycytidilic acidcomplexed with poly-L-lysine and carboxymethylcelulose. Research inVeterinary Science 44:132-133). Pigs in both groups were given asecondary “booster” immunization 8 weeks following primary immunizationthat included poly lCLC for the appropriate group. Peripheral bloodmononuclear cells were isolated weekly and used to monitor thevirus-specific immune response to the PRRS virus vaccine. The number ofPRRS virus-specific IFN-gamma-secreting cells was measured using anELISPOT assay as described (Zuckermann et al. 1998).

The accompanying figure (FIG. 4) shows the enhancing effect of polylCLCon the virus-specific IFN-gamma response to vaccination against PRRSV.Groups of pigs (n=5) were immunized with a commercial PRRSV modifiedlive virus vaccine given either alone (open squares) or in combinationwith the simultaneous intramuscular injection at an adjacent site ofpolylCLC at a dose of 0.25 mg/kg of body weight (open diamonds). At theindicated times peripheral blood mononuclear cells were isolated fromthese animals and tested in an IFN-gamma ELISPOT assay as described(Zuckermann et al., 1998). The IFN-gamma response is expressed as thegroup mean frequency of virus-specific IFN-g-secreting cells/10⁶PBMC±standard error of the mean.

Example 5 Studies of an Inactivated, Subunit, or Attenuated PRRS VaccineComprising IFN″ cDNA Adjuvant

In this example, circumstances are similar to that for the examples withthe PRRS MLV vaccine (with or without challenge), but the PRRS vaccinehere comprises either an inactivated form of PRRS or comprises a subunitof PRRS, such as a polypeptide. Alternatively, the PRRS vaccinecomprises an attenuated form of PRRS that is attenuated by a processother than that corresponding to the process for the PRRS MLVspecifically described as the Ingelvac type. The process of attenuationcan be achieved by methods known in the art such as passage in vitro,passage in foreign hosts, or by methods including genetic engineering. Anon-limiting example of such an attenuated strain can be derived fromATCC VR-2385.

Any examples provided are not intended as exclusive or limiting; thescope of the claims is intended to be determined by the claim languageand not by specific examples or embodiments listed. All references citedherein are incorporated in their entirety by reference herein to theextent that they are not inconsistent with the disclosure herein.

Any sequence listing information, whether submitted in paper orelectronic form, including any submission via the USPTO EFS-WEB systemas a *.txt file or other format, is part of the specification herein.

1. An adjuvant composition comprising a nucleic acid capable ofexpressing an interferon alpha in a pig in an amount sufficient toenhance an immune response to a PRRS virus when administered to the pig.2. The adjuvant composition of claim 1 wherein the nucleic acidcomprises the sequence of SEQ ID NO:3.
 3. A method for enhancing animmune response of an animal to a vaccine comprising the steps of: a.administering a PRRS vaccine antigen composition to an animal in anamount effective to stimulate an immune response; and b. administeringto the animal an adjuvant composition comprising a nucleic acid capableof expressing interferon alpha in an amount effective to potentiate theimmune response to the vaccine antigen composition.
 4. The method ofclaim 3 wherein the interferon alpha is a porcine interferon α.
 5. Themethod of claim 3 wherein said animal is a non-human mammal.
 6. Themethod of claim 3 wherein said animal is a pig.
 7. The method of claim 3wherein said vaccine antigen composition comprises a vaccine antigenselected from the group consisting of live vaccine virus, inactivatedvaccine virus, subunit vaccine, and polypeptide vaccine.
 8. The methodof claim 7 wherein said live vaccine virus is derived from an agentcapable of causing clinical signs of porcine reproductive andrespiratory syndrome disease.
 9. The method of claim 7 wherein said livevaccine virus is derived from ATCC-VR2495.
 11. The method of claim 3wherein said nucleic acid comprises SEQ ID NO:3.
 12. The method of claim3 wherein said administering steps are independently by a type ofadministration selected from the group consisting of: parenteralinjection, intravenous injection, intranasal inoculation, per osinoculation, intraocular inoculation, inhalation inoculation,transmucosal inoculation, and transdermal inoculation.
 13. The method ofclaim 12 wherein said administration steps occur by the same ordifferent types of administration.
 14. The method of claim 12 whereinsaid administration steps are concurrent or sequential.
 15. The methodof claim 12 wherein said administration of said adjuvant occurs from oneweek before to one week after said administration of said vaccine. 16.The method of claim 12 wherein said vaccine antigen compositioncomprises a live vaccine virus, further comprising at least onesubsequent administration of the live vaccine virus.
 17. The method ofclaim 12 further comprising at least one subsequent administration ofsaid nucleic acid.
 18. A composition comprising an effective amount of aPRRS vaccine antigen composition and an effective amount of an adjuvantwherein the adjuvant is a nucleic acid capable of expressing interferonα, wherein the amount of the adjuvant is sufficient to enhance theanimal's immune response to the vaccine and wherein the vaccine antigenis present in an amount sufficient to raise an immune response to theantigen.
 19. The composition of claim 18 wherein said vaccine antigencomposition comprises a vaccine antigen selected from the groupconsisting of live vaccine virus, inactivated vaccine virus, subunitvaccine, and polypeptide vaccine.
 20. The composition of claim 19wherein said live vaccine virus is derived from an agent capable ofcausing clinical signs of porcine reproductive and respiratory syndromedisease.
 21. The composition of claim 19 wherein said live vaccine virusis derived from ATCC-VR2495.
 22. The composition of claim 18 whereinsaid nucleic acid is capable of expressing porcine interferon α.